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Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis.


ABSTRACT: Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade beta-propeller fold linked to a C-terminal beta-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (-1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the -1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.

SUBMITTER: Chuankhayan P 

PROVIDER: S-EPMC2906318 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis.

Chuankhayan Phimonphan P   Hsieh Chih-Yu CY   Huang Yen-Chieh YC   Hsieh Yi-You YY   Guan Hong-Hsiang HH   Hsieh Yin-Cheng YC   Tien Yueh-Chu YC   Chen Chung-De CD   Chiang Chien-Min CM   Chen Chun-Jung CJ  

The Journal of biological chemistry 20100513 30


Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raf  ...[more]

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